Pharmacology & Pharmacy, 2011, 2, 136-140
doi:10.4236/pp.2011.23018 Published Online July 2011 (http://www.scirp.org/journal/pp)
Chromatographic Separation and
Characterization of Capsaicinoids and Their
Analogues*
Junlian Wang1, Li Zhou2, Lei Wang2, Zhenghong Peng3, Shengze Zhou4, Xiangfeng Zhou5,
Xiaobin Zhang1, Bixian Peng6#
1Department of Materials Science and Engineering, Zhejiang University, Hangzhou, China; 2Chenguang Engineering Technology
Research Center of Natural Pigments, Hebei, China; 3Beijing TianGongLongYuan International Biochemical Company, Beijing,
China; 4Beijing JiuZhouLongLing Environmental Protection Material Company, Beijing, China; 5Institute of Chemistry, Chinese
Academy of Sciences (CAS), Beijing, China; 6Technical Institute of Physics and Chemistry, CAS, Beijing, China.
Received December 19th, 2010; revised May 4th, 2011; accepted May 15th, 2011.
ABSTRACT
An attempt was made to establish a chromato graphic separation and analysis method for capsaicinoids and their ana-
logues. A series of factors,such as choice of column and eluents, elu ent composition ,flow rate of eluent and operation al
temperature were correlated, countereacted and optimized to attain appropriate separation efficiency and finalize the
experimentally oprational procedures as a whole, permiting a mixture comprising 8 capsaicinoids including analogues
to be separated and Characterized with an allowed deviations and within a period of 36 minutes via 1 test as well.
Keywords: Capsaicinoid, Chromatography, Separation, Characterization
1. Introduction
In our previous paper [1], it has been introduced that
capsaicin had been effectuated to have shown mutagenic,
anti-proliferative and anticarcinogenic properties toward
human prostate gland’s, lung cancer’s and leukemic cells
[2-10] and highlighted that capsaicinoids differ from
each other in pungency depending on the molecular con-
crete structure involved, leading to a conclusion that
capsaicin and dihydrocapsaicin had been ranked to be the
most pungent ones. Combining the research results
originated and come from completely different 2 sectors-
one is the curative effect of capsaicin displayed in phar-
macology with respect to different cancer cells, the other
is pungency structure dependence exploited in bioor-
ganic chemistry-as a whole, it seems to us to have seen
what a bit light of early dawn and we are hopefully
imagined that the potential biologically curative value
and even the possible clinical administration and obser-
vation of capsaicinoid deserve attention and concerns of
medical researchers in deed. In order to bolster such a
kind of systematic research, first of all, it is necessary to
prepare capsaicinoids, firstly the capsaicin and dihydro-
capsaicin, in sufficient high quality and in ample quanti-
ties as to undertake a systematic as well as multifactorial
studies from different angles and points of view. To our
best knowledge there are two principle routes, one is
traditional by which the capsaicin and dihydrocapsaicin
is separated and manufactured from the extracts ex-
tracted from the dry powders of hot peppers whose na-
tive texture contains capsaicin, dihydrocapsaicin, nordi-
hydrocapsaicin, homocapsaicin, homodihydrocapsaicin
etc., the other is artificial with the help of which individ-
ual capsaicinoid, such as capsaicin, dihydrocapsaicin can
be synthesized by reacting vanillylamine and the corre-
sponding acid chloride as outlined in Equation (1) of our
previous article [1]. As far as the traditional route is con-
cerned, it is advantageous and featured by the case of
raw material available for massive production, the safety
as drugs to be administrated to the patients and the plenty
in supply. However anything in the world is twofold. The
traditional route is likewise disadvantageous and charac-
terized by a complexity of the technological production
*Capsaicinoids are referred to all the pungent members of capsaicin
family present in natural hot pepper. Analogues are referred to those
artificial pungent compounds whose molecular structures are similar to
capsaicinoids but unable to be detected in native hot pepper.

Chromatographic Separation and Characterization of Capsaicinoids and Their Analogues 137
line, long term period of recycle and the extremely high
price of the final products. In a word as compared with
the traditional route, the artificial one had displayed a
few distinguished superiorities , such as a comparatively
shortened and reduced preparation and separation pro-
cedures and much lower production cost of the products.
Based on our experience and practice, all routes,
whether they are traditional, artificial even or enzymatic
as well [11], are required to have a liquid chroma-
tographic analytical method able to separate and charac-
terize the possible analogues of capsaicin or dihydrocap-
saicin originated either from the natural raw materials in
case of traditional procedures, or from the sub-standard
chemical reagent raw material (<95% purity , for exam-
ple, isodecyl alcohol with minor impurities of iso-nonyl
and iso-octyl alcohols, <5%) in case of artificial proce-
dures for dihydrocapsaicin-making.
A survey and investigation of literature enables us to
be aware of a fact that neither published papers nor ap-
proved patents can provide a method or a technology by
which a good many (8 - 10) of capsaicinoids or artificial
analogues of capsaicin and dihydrocapsaicin can be sepa-
rated and characterized by 1 run of chromatographic test
within a short period less than 40 minutes.
A required chromatographically analytical method
comprising column, detector, fixed phase, flow phase etc.
is addressed to separate the possible capsaicinoids such
as capsaicin, dihydrocapsaicin, nordihydrocapsaicin, ho-
mocapsaicin and the artificial capsaicin analogues as
listed in Tab le 1, at which is the right purpose of present
study aimed, presented and discussed.
2. Experiment
2.1. Analogues
The chemical name, chemical formula of 8 synthesized
analogues of capsaicin and dihydrocapsaicin in our labo-
ratory according to the procedures outlined in paper [1]
used for chromatographic separation and analysis are
listed in Table 1.
2.2. Analysis
2.2.1. Re a gent
Acetonitrile, chromatographic grade; Glacial acetic acid,
99.5%, analytical grade; Ethanol, analytical grade manu-
factured by Handan Fine Chemical Company, Province
Hebei, China.
2.2.2. Sampl e S ol ution
Approximately 0.016 g of each analogue sample is
weighed precisely and dissolved in ethanol (100 ml),
from which an aliquot (3 ml) was sucked out to form the
analogues mixture stock solution. 20 μl of the said stock
solution, after passing through a filter membrane (0.45 μ
pore diameter), was injected into the column used.
2.2.3 Seeking for Optimum Separation Procedure
Apparatus
Liquid Chromatography Agileng 1200, Chromatographic
Column A.E. Lichyon, C-18, 5 μm, two columns differ
in length, one is 150 mm, the other 250 mm respectively.
Flow phase
1-st flow phase is methanol plus H2O, whose relative
percentage (%) is prepared to be (methanol: H2O) 50:50,
60:40, 65:35, 70:30 and 75:25.
The 2-nd flow phase is acetonitrile: H2O: glacial acetic
acid whose percentage (%) is consisted of 30:70:0.6,
40:60:0.6, 45:55:0.6 and 50:50:0.6 respectively.
A common normalized conditions (column C-18, 250
mm long, column temperature at 30˚C and the constant
flow rate 1.5 ml/min) is used in an effort to seek for the
optimum flow phase’s experimental conditions beneficial
to improve the separation results
3. Results and Discussion
3.1. Optimization of Analytical Conditions
Column
Prolongation of the column length from 150 mm to
250 mm has brought about an extention of 1 run from 42
minutes to 75 minutes, however created no remarkable
improved separation efficiency, in particular for the ana-
logues with much more closer molecular structures (No.
2, 3, 4 and 5 in Table 1). A shorter column (150 mm) is
preferable for use.
Flow phase
A comparative study of a same solution but with dif-
ferent percentage (%) proportions showed that the solu-
tion with the percentage proportion of ethanol: H2O
(75:25) had a better promotion effect with regard to bet-
tering a separation efficiency.
A similar promotion effect is also with the ternary
system of acetonitrile: H2O: glacial acetic acid with per-
centtage proportion (45:55:0.6). In order to save space all
the 9 chromatogram maps for above 9 proportions (%)
are omitted.
Elution rate
An experimental attempt was made to watch in which
degree an deviation from an usual elution rate of 1.5
ml/min can be affected. Neither a deviation change from
1.5 ml/min to a smaller rate, such as 0.85 ml/min, nor to
a longer rate, for example, to 2.0 ml/min could brought
about a bit improvement. A middle rate is the best of 1.5
ml/min.
From the above-mentioned comparative studies it per-
mits to make a fundamental conclusion that an optimum
xperimental conditions for chromatographic separation e

Chromatographic Separation and Characterization of Capsaicinoids and Their Analogues 138
Table 1. The structure, retention time (RT), peak area and percentage content (%) of capsaicinoids and analogues.
No. Structure RTs RTm A
x %
1 14.34 14.25 198.4 11.24
2
22.01 21.87 39.8 2.26
3 12.97 12.91 75.46 4.28
4 11.20 11.15 734.3 41.60
5 15.06 14.99 130.8 7.41
6
38.28 38.50 99.8 5.66
7 13.67 13.59 237.7 13.47
8 16.75 16.68 249.2 14.12
RTs: retention time for single sample injection; RTm: for mixture sample injection.
of analogues of capsaicin and (or) dihydrocapsaicin can
be recommended as follows: column C-18, 150 × 4.6
mm, 5 μm, flow phase acetonitrile-H2O-glacial acetic
acid in percentage proportion 45.0:54.4:0.6; flow rate of
eluent 1.5 ml/min and a temperature of column 30˚C.
Under the optimized conditions each individual ana-
logues’s solution (20 μl) was injected alone and the spe-
cific retention time for quantitative analysis was found.
The experimentally found retention time for all the 8
analogues was determined and summarized in the figure
note of Figure 1. From Figure 1 it can be seen that the
retention time of each individual analogue obtained by
single analogue injection (No. 1-8) are in very good co-
incidence with that found by mixture analogue ingection
within an allowed deviations and that among the 8 ana-
logues (No. 1-8 in Table 1) 7 analogues are separated
and characterized quite well with an unique fault that
howed an overlapping of the retention time of No. 7with s

Chromatographic Separation and Characterization of Capsaicinoids and Their Analogues 139
Figure 1. Chromatogram map for separation of No. 1-5 and No. 7-8.
Figure 2. Chromatogram map for separation of No. 1-8.
that of No. 6. In order to avoid and overcome this fault a
fine tuning of the acidity of the ternary eluent system
was made and the 8 analogues’ mixture was basically
separated and quantitatively determined without over-
lapping peaks like No. 6 and No. 7 (Figure 2).
The establishment of chromatographic separation and
characterization is of practical significance. Firstly for
analysis of natural capsaicinoids, in principle, applied

140 Chromatographic Separation and Characterization of Capsaicinoids and Their Analogues
with minor modification, the analyzing scope of capsai-
cinoids may be expanded to the minor members of cap-
saicinoid, such as homohomocapsaicin and nornordi-
hydrocapsaicin etc. whose content in native hot pepper
amount totally less than 2% - 3%; Secondly for analysis
of artificial analogues including those pungent com-
pounds with similar or approximate structures to each
other that are not existed in native hot pepper but are able
to be synthesized in chemical laboratory. It is believed
that the recommended chromatographic procedure may
promote the preparative progress of high-qualified
capsaicinoids and their analogues that hold promise of
being candidate drugs for a potential anticarcinogenic
use.
3.2. Summary
1) An optimized Chromatographically operational
conditions for a apsaicinoids’ mixture separation can be
summarized as follows: column C-18, 250 × 4.6 mm. 5u,
flow phase acetonitrile -H2O- glacial acetic acid in per-
centage (V/V) 45.0; 54.4:0.6, flow rate of eluent 1.5
ml/min and a column temperature at 30˚C.
2) 8 capsaicinoids/analogues are able to be separated
and characterized with an allowed deviations and within
a short period of 36 munites via 1 chromatographic ex-
periment.
3) The suggested chromatographic analytical method
holds promise to be used in principle for separating and
characterizing native capsaicinoids as well as artificial
analogues for medical usage.
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